The long-term goal of this research is to further develop and validate a new class of small molecule enzyme sensing, near infrared fluorescence (NIRF) reporter probes for in vivo imaging. We believe that such "smart agents" could be essential in imaging a wider spectrum of disease processes. Our laboratory has developed several different enzyme sensing probes, such as cathepsin B, cathepsin D, matrix metalloprotease (MMP)-2 and thrombin sensitive probes, all based on long circulating graft copolymers (> 450 kD) bearing protease sensing peptide stalks with quenchable fluorochrome reporters. Although these high molecular weight probes have been used in investigating different biological questions, probes with smaller molecular weights would have several advantages for imaging: a) faster tissue distribution allowing earlier imaging after injection, b) faster clearance allowing repeated imaging, c) higher likelihood of intracellular delivery and d) higher likelihood of developing key candidates into clinically useful agents. Such enzyme activatable low molecular weight imaging probes have unique design issues requiring a) bright and narrow spectral width of the near infrared fluorochromes and b) efficient NIRF quenching molecules. In preliminary work we have already developed a platform of tightly tuned NIRF fluorochromes and have also designed highly efficient quenchers based on azulene structures. Using these developed compounds we propose to optimize, characterize and investigate three different sensors, capable of imaging matrix metalloprotease (MMP)-7, MMP-9 and MMP-13. The choice of these targets is based on their importance in angiogenesis, tumor invasion and other processes in oncogenesis. Together with recent developments in tomographic fluorescence imaging technologies, this research is expected to ultimately result in clinical imaging agents with specificity for targeted proteases (and potentially other enzymes) playing key roles in tumor progression and other diseases.